(a) Field of the Invention
The invention relates to compounds which exhibit anti-KS and anti-HIV activity, pharmaceutical compositions and method of treatment thereof.
(b) Description of Prior Art
Kaposi""s sarcoma (KS) is the most common tumour in AIDS subjects which afflicts high mortality (Friedman-Kien A E et al., 1990, J Am Acad Dermatol 22:1237-1250). Less aggressive forms can also occur in non-AIDS subjects of the Mediterranean area and equatorial Africa as well as in renal transplant patients following treatment with immunosuppressive drugs (Friedman-Kien A E et al., 1990, J Am Acad Dermatol 22:1237-1250). The pathogenesis and therapy of KS remain enigmatic (Bais C. et al., 1998, Nature 391:86). For unknown reasons, occurrence of KS is higher in males than in females. For example, in the West, approximately 95% of AIDS-KS subjects are men. Although, hormonal dependence of KS has been demonstrated in the case of glucocorticoid and retinoid (Guo W X et al., 1996, Am J Pathol 148: 1999-2008; Guo W X et al., 1995, Am J Pathol 146: 727-734; Guo W X et al., 1995, Cancer Res 55: 823-829), sex steroids do not seem to be directly involved in KS pathogenesis. Recently, Lunardi-Iskandar et al. (Lunardi-Iskandar Y et al., 1995, Nature 375: 64-68) reported that the placental hormone human chorionic gonadotropin (HCG), displays anti-KS activity and prevents tumours in immunodeficient mice. This preliminary finding could otentially have significant therapeutic impact as demonstrated in clinical trials (Gill PS et al., 1996, New Engl J Med 335: 1261-1310; Gill P S et al., 1997, J. Natl. Cancer Inst. 89: 1797) and may shed light on basic understanding of this disease particularly regarding the sexual dimorphism issue. Though the role of HCG is principally to sustain pregnancy, it is becoming increasingly apparent that this hormone, possibly along with other active molecules, may be responsible for numerous other phenomena. The low transmission rate of HIV across the placenta (Prober C G et al., 1991, Ped Infect Dis J 10: 684-695) as well as the low incidence of Kaposi Sarcoma in women including those previously infected with the virus has led to the suspicion that pregnancy and/or reproductive hormones (such as related LH, see Lunardi-Iskandar Y et al., 1995, Nature 377: 21-22) may be involved in curtailing the propagation of the virus. Studies by Bourinbaiar (Bourinbaiar AS et al., 1995, Immunol Lett 44: 13-18) indicate that the hormone HCG or its xcex2 subunit may have an anti-HIV effect. The action of HCG on gonadal cells is mediated by a G-protein coupled trans-membrane receptor which interacts with the dimeric hormone (xcex1 and xcex2 complex) with very high affinity and specificity (review Segaloff D L et al., 1993, Endocr Rev 14: 324-347). In such a system, it is very well known that either one of the individual xcex1 and xcex2 subunits have extremely low reactivity towards the membrane bound receptor (Pierce J G et al., 1981, Ann Rev Biochem 50: 465-495; Sairam M R, 1983, In: Hormonal Proteins and Peptides. Li C. H., ed., pages 1-79) but complete activity can be regained by appropriate (1:1) recombination of the two subunits. Lunardi-Iskandar""s and Bourinbaiar""s data (Lunardi-Iskandar Y et al., 1995, Nature 375: 64-68; Bourinbaiar A S et al., 1995, Immunol Lett 44: 13-18) suggest the involvement of an xe2x80x9cunconventionalxe2x80x9d mode of action for HCG in KS. In fact, they reported a biological activity for the xcex2 HCG, a notion which contradicts the generally accepted paradigm that the dimeric form of the hormone is required for triggering hormonal responses in classical target tissues. While stirring a controversy (Lunardi-Iskandar Y et al., 1995, Nature 377: 21-22; Berger P et al., 1995, Nature 377: 21; Rabkin C S et al., 1995, Nature 377: 21-22; Krown S E, 1996, New Engl J Med 335: 1309-1310), these findings raise intriguing and potentially novel issues.
There is reported in Nature Medicine (Vol. 4, No. 7, July 1998) that the anti-KS activity of crude hCG preparations is still a mystery.
It would be highly desirable to be provided with compounds which would exhibit anti-KS and anti-HIV activity.
One aim of the present invention is to provide with compounds which would exhibit anti-KS and anti-HIV activity.
In accordance with one preferred embodiment of the present invention there is provided a compound having anti-KS and anti-HIV pharmaceutical activity which comprises an RCG-like inhibitory protein and fragments thereof, the protein and fragments thereof are isolated from a biologically active fraction of APL(trademark)-HCG (xe2x80x9cAPL(trademark)xe2x80x9d is the commercial trade name of the clinical-grade HCG sold by Wyeth-Ayerst), wherein said protein has a molecular weight of about 3,500 or of about 13,000 Dalton, and wherein said protein and fragments thereof are adsorbed to polypropylene plastic supports, such as tubes or pipette tips among others.
A preferred polypropylene plastic tube includes those sold by Sarstedt (Numbreht, Germany) cat #57.512 and cat #68.752.
In accordance with another preferred embodiment of the present invention there is provided purified protein and derivatives and fragments thereof having anti-KS and anti-HIV pharmaceutical activity which is a HCG-like inhibitory protein and derivatives and fragments thereof which are adsorbed to polypropylene plastic supports, and wherein said protein has an amino acid sequence selected from the group consisting of:
Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Glu-Arg-Pro-Ile-Asn*-Ala-Thr-Leu-Ala-Val-Glu-Lys SEQ ID NO:1; and
Ala-Pro-Asp-Val-Gln-Asp-Lys-Phe-Thr-Arg-Gln-Ile-Met-Ala-Thr SEQ ID NO:2.
The purified protein of the present invention is referred to as HIP or HCG-like Inhibitory Protein.
In other embodiments, the derivatives contain one or more D-amino acids or non-natural amino acids.
In accordance with another preferred embodiment of the present invention there, is provided a pharmaceutical composition for the prevention and/or treatment of Kaposi""s sarcoma (KS) and/or HIV which comprises a therapeutically effective amount of at least one compound of the present invention in association with a pharmaceutically acceptable carrier.
In accordance with another preferred embodiment of the present invention there is provided a pharmaceutical composition for the prevention and/or treatment of Kaposi""s sarcoma (KS) and/or HIV which comprises a therapeutically effective amount of at least one protein of the present invention in association with a pharmaceutically acceptable carrier.
In other embodiments, the pharmaceutical composition is formulated as a controlled release formulation.
In accordance with another preferred embodiment of the present invention there is provided a pharmaceutical composition for the prevention and/or treatment of Kaposils sarcoma (KS) and/or HIV which comprises a therapeutically effective amount of a derivative of a protein having anti-KS and anti-HIV pharmaceutical activity which is a HCG-like inhibitory protein which is adsorbed to polypropylene plastic supports, and wherein said protein has an amino acid sequence selected from the group consisting of:
Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Glu-Arg-Pro-Ile-Asn*-Ala-Thr-Leu-Ala-Val-Glu-Lys SEQ ID NO:1; and
Ala-Pro-Asp-Val-Gln-Asp-Lys-Phe-Thr-Arg-Gln-Ile-Met-Ala-Thr SEQ ID NO:2
in association with a pharmaceutically acceptable carrier.
In accordance with another preferred embodiment of the present invention there is provided a method for the prevention, treatment and/or reduction of Kaposi""s sarcoma and/or HIV expression in AIDS patients, which comprises administering to said patient a therapeutically effective amount of a compound of the present invention.
In accordance with another preferred embodiment of the present invention there is provided a method for the prevention, treatment and/or reduction of Kaposi""s sarcoma and/or HIV expression in AIDS patients, which comprises administering to said patient a therapeutically effective amount of a protein of the present invention.
In accordance with another preferred embodiment of the present invention there is provided a method for the prevention, treatment and/or reduction of Kaposi""s sarcoma and/or HIV expression in AIDS patients, which comprises administering to said patient a therapeutically effective amount of a pharmaceutical composition of the present invention.
In accordance with another preferred embodiment of the present invention there is provided a method to purify the compound or protein of the present invention, which comprises the steps of:
a) subjecting a biologically active fraction of APL-HCG or urinary extract containing said compound or protein to a polypropylene plastic support for a time sufficient for adsorption of said compound or protein to occur; and
b) washing the support and releasing the adsorbed compound or protein therefrom.
In accordance with another preferred embodiment of the present invention there is provided a method of evaluating inhibitory activity of anti-KS and anti-HIV compound, which comprises by measuring AP1 gene activity.
In other embodiments, measuring of said AP1 gene activity is effected by measuring binding to DNA response element.
For the purpose of the present invention the following terms are defined below.
xe2x80x9cHIPxe2x80x9d: HCG-like Inhibitory Protein;
xe2x80x9cHPLCxe2x80x9d: high-pressure liquid chromatography; and
xe2x80x9cAPLxe2x80x9d: commercial trade name of the clinical-grade HCG sold by Wyeth-Ayerst, cat. #DIN 02168936.
The expression xe2x80x9cderivatives and fragments thereofxe2x80x9d is intended to mean any derivatives and fragments of the protein of the present invention which exhibit anti-KS and anti-HIV pharmaceutical activity effective for the prevention, treatment and/or reduction of Kaposi""s sarcoma in AIDS patients. The derivatives may include one or more D-amino acids or non-natural amino acids. The derivatives and fragments are functional and substantially exhibit the biological activity of the protein of the present invention.